WO2024044319A2 - Matériau de dentine biomimétique et réparateur pour favoriser la régénération de tissu de dentine et procédés associés - Google Patents

Matériau de dentine biomimétique et réparateur pour favoriser la régénération de tissu de dentine et procédés associés Download PDF

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WO2024044319A2
WO2024044319A2 PCT/US2023/031056 US2023031056W WO2024044319A2 WO 2024044319 A2 WO2024044319 A2 WO 2024044319A2 US 2023031056 W US2023031056 W US 2023031056W WO 2024044319 A2 WO2024044319 A2 WO 2024044319A2
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Prior art keywords
dentin
pillars
tubular recesses
bioartificial
matrix
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PCT/US2023/031056
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English (en)
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WO2024044319A3 (fr
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May Anny Alves FRAGA
Luiz E. BERTASSONI
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Oregon Health & Science University
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Publication of WO2024044319A3 publication Critical patent/WO2024044319A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K6/00—Preparations for dentistry
    • A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
    • A61K6/831—Preparations for artificial teeth, for filling teeth or for capping teeth comprising non-metallic elements or compounds thereof, e.g. carbon
    • A61K6/838—Phosphorus compounds, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane

Definitions

  • the present disclosure relates generally to regenerative dentistry. More specifically, the present disclosure relates to materials and methods for promoting the regeneration of dental tissue.
  • Dentin is the largest structure in the tooth, and is composed of hydroxyapatite mineral crystallites, collagen fibrils (mostly type I) and non-collagenous molecules.
  • odontoblasts secrete dentin matrix in a well-orchestrated sequence, characterized by the release of collagen fibrils associated with high concentrations of carboxylated non-collagenous proteins and proteoglycans.
  • These dentin matrix organization and constituents regulate the process of mineral deposition and fibrillogenesis.
  • Subsequent phase transformations allow the amorphous mineral to adopt a more crystalline and stable hydroxyapatite morphology and form an intra- and extrafibrillar mineral, which is crucial for the tooth function.
  • intrafibri liar mineralization is critical for dentin's mechanical and biological properties.
  • odontoblasts are stimulated to secrete organized tubular dentin to form a calcified tissue separating the dental pulp from the challenging oral environment. This tissue is known as reactionary dentin.
  • deeper cavities, or intense stimuli detrimental to dental pulp cells result in death of odontoblasts triggering a strong inflammatory response, followed by the recruitment of stem and progenitor cells which differentiate into odontoblasts and secret a less organized and denser dentin.
  • This tertiary dentin is "more protective" due to the lower diffusion rate of soluble byproducts through the tubule-less tissue.
  • the gold standard materials for vital pulp therapy are cement-based materials that lack the complexity of the dentin and have their biological response based on triggering an inflammatory response in an already injured dental pulp that will eventually result in the secretion of reparative dentin. This process is slow and the high inflammation makes it far from ideal.
  • FIG. 1 is a schematic showing mineral trioxide aggregate (MTA) causing severe inflammation, while eDentin is a 100% biocompatible and non-inflammatory, ready-to-use membrane that is placed onto the dental pulp (like a “band-aid”) releasing dentin matrix molecules (DMMs) to attract pulp cells that will secrete fresh calcified matrix, thus integrating eDentin into the pulp — dentin complex.
  • MTA mineral trioxide aggregate
  • DDMMs dentin matrix molecules
  • Figures 2A to 2G are images and bar graphs showing SEM and TEM of eDentin’s nanostructure vs. native calcified collagen fibril (FIGS. 2A-2D); mineralization, composition, and elastic modulus of eDentin vs. pure collagen and native mineralization tissue controls (FIGS. 2E-2G).
  • Figures 3A to 3G are images of SEM before (FIG. 3A) and after (FIG. 3B) condensation; eDentin can be held with tweezers (FIG. 3C); viability (FIG. 3D) and morphology (FIG. 3E) of embedded cells; eDentin outside and inside the tooth (FIGS. 3F-3G).
  • FIG. 4A proteomic percentage of extracted dentin matrix molecules (DMMs);
  • FIG. 4B tertiary dentin formation using engineered hydrogels supplemented with DMMs; and
  • FIG. 4C MTA induced inflammation prior to tertiary dentin formation in-vivo.
  • FIGs 5A to 5D are images of Rat’s molars having had their pulp exposed and treated with (FIG. 5B) MTA or (FIG. 5C) eDentin;
  • FIG. 5A is a MicroCT scan showing mineralized eDentin capping the dental pulp;
  • FIG. 5B histological sections of MTA samples showing tissue disorganization, and severe inflammation (arrows), while eDentin (FIG. 50, 5D - asterisk) is not inflammatory, can be immediately juxtaposed with the pulp tissue (FIG. 5D - dotted line) as a ready-to-use mineralized engineered dentin (i); H&E and von Kossa.
  • Figures 6A to 6C shows vvaluation regarding the order of use of the crosslinker relative to mineralization.
  • Figures 6A to 6C show that mineralization can be performed either prior to or following crosslinking of the collagen.
  • FIG. 6A and FIG. 6B show better mineralization is achieved prior to crosslinking as evidenced by higher mineral to matrix ratio and crystallinity index from FTIR data as well as the more opaque appearance in photographs.
  • FIG. 7A shows scanning Electron Microscope (SEM) images demonstrate the surface morphology with different crosslinkers appear comparable.
  • FIG. 7B shows cell viability, as determined via a cell metabolic activity MTT assay, appear comparable using different crosslinking agents.
  • SEM scanning Electron Microscope
  • FIG. 7B shows cell viability, as determined via a cell metabolic activity MTT assay, appear comparable using different crosslinking agents.
  • mineralization was performed prior to crosslinking. No difference in cell viability (cytotoxicity) observed among different crosslinking agents.
  • FIG. 8A to 8G shows the characterization of mineralized collagen.
  • FIG. 8E EDX spectra of high-density mineralized samples confirmed the presence of Ca and P.
  • FIG. 8F mineral: matrix ratio and
  • FIG. 9 shows DMM release kinetics confirms that DMMs are incorporated and released from eDentin.
  • DMM Release kinetics from eDentin into media with up to 80% release in the first 48 hours confirms that DMMs are both incorporated and released from eDentin.
  • the DMM release protein assay was done in different timepoints (1 h, 12h, 1day, 2 days and 7 days) using BCA Protein Assay Kit.
  • Figures 10A to 10B shows eDentin (compressed, mineralized high-density fibrillar collagen with DMM, abbreviated cm-HDFC + DMM) supports better mineral deposition by odontoblast like cells (OD-21 cells) compared to high-density fibrillar collagen (HDFC) alone using a Alizarin red S stain.
  • FIG. 10A imagens
  • FIG. 10B Quantification of Alizarin red S stain.
  • FIG. 11 A Scanning electron microscope image showing that eDentin is able to bond to formulated adhesive containing Methacryloyloxydecyl dihydrogen phosphate (MDP) and hydrophilic photoinitiator.
  • MDP Methacryloyloxydecyl dihydrogen phosphate
  • FIG. 11 B 10- MDP and hydrophilic photoinitiator after shear stress. The collagen is present on the surface suggesting a bond between the adhesive and the membrane.
  • FIG. 11C Adhesive single bond plus after sheer stress. There is less collagen present on the surface suggesting the adhesion is not good.
  • FIG 12 is a schematic showing an exemplary application of eDentin in pulp capping procedures.
  • An adhesive containing 10-MDP and hydrophilic photoinitiator can be applied over the eDentin surface as shown in D.
  • the 10-MDP monomer is capable of chemically bonding to calcium present in the membrane as shown in D. and the resin matrix in the restorative material as shown in E.
  • Hydrophilic photoinitiator is important to promote the monomer conversion in a wet surface (look the last figure).
  • the application mode of eDentin for glass ionomer is after cavity preparation where the eDentin must be inserted over the exposed pulp. After eDentin application, the glass ionomer can be applied according to the manufacture instructions.
  • collagenous material refers to a composition comprising collagen.
  • biomimetic refers to a material engineered to have qualities or properties that mimic (i.e., resemble or imitate) the qualities or properties of a native tissue.
  • a reparative dentin-like biomaterial is configured to mimic the biomimetic nanometer scale structural properties of native dentin tissue such as a dense and calcified collagenous matrix, a tubular micromorphology, and the presence of dentin-derived matrix molecules and embedded, differentiated stem/progenitor cells expressing a mineralizing phenotype.
  • collagen refers to any form of a collagen, such as a type 1 collagen matrix.
  • a type 1 collagen matrix may be prepared by reconstituting acid solubilized type 1 collagen.
  • the collagen may be at a concentration of about 0.5 mg/mL to about 100 mg/mL.
  • collagen examples include: collagen type II, collagen type III, collagen type IV, collagen type V, collagen type VI, collagen type VI, collagen type VII, collagen type VIII, collagen type IX, collagen type X, collagen type XI, collagen type XII, collagen type XIII, collagen type XIV, collagen type XV, collagen type XVI, collagen type XVII, collagen type XVIII, collagen type XIX, and collagen type XX, or a combination thereof.
  • Type I collagen” or “Type 1 collagen” refers to the fibrillar-type collagen that is the most abundant form of human collagen and the key structural composition of several tissues.
  • DPSC dental pulp stem cells
  • SHED exfoliated deciduous teeth
  • DFPC dental follicle precursor cells
  • PDLSC periodontal ligament stem cells
  • SCAP gingiva-derived mesenchymal stem cells
  • DMM dense matrix molecules
  • DSPP dentin sialophosphoprotein
  • DMP-1 dentin matrix protein-1
  • BSP BSP, OPN
  • MEPE phosphorylated matrix proteins
  • MMP-1 collagenase
  • MMP-2 gelatinases
  • MMP-9 stromelysin
  • MMP- 3 enamelysin (MMP-20)
  • MT1-MMP TIMP-1, TIMP-2, TIMP-3
  • alkaline phosphatase serum-derived proteins aHS2-glyco
  • DMMs are provided by Bertassoni et al., Archives of Oral Biology, 119 (2020) 104888), which is hereby incorporated by reference in its entirety. For example, as disclosed herein, it was observed that application of DMMs to a repair site promoted the migration and differentiation of host stem cells to the repair site.
  • dentin matrix protein refers to any of the proteins or glycoproteins present in native dentin, particularly including, but not limited to, dentin matrix acidic phosphoprotein 1 (DMP1 ), dentin phosphophoryn (DMP2), dentin matrix protein 4 (DMP4 or FAM20C), dentin sialophosphoprotein (DSP), bone sialoprotein, osteopontin, osteonectin, osteocalcin, osteopontin, decorin, and matrix extracellular phosphoglycoprotein (MEPE).
  • DMP1 dentin matrix acidic phosphoprotein 1
  • DMP2 dentin phosphophoryn
  • DMP4 or FAM20C dentin matrix protein 4
  • DSP dentin sialophosphoprotein
  • bone sialoprotein bone sialoprotein
  • osteopontin osteonectin
  • osteocalcin osteopontin
  • decorin matrix extracellular phosphoglycoprotein
  • dentin-derived matrix molecules refers to dentin matrix molecules that have been derived from one or more samples of natural dentin or pulp from one or more mammalian subjects.
  • tissue tubule refers to one of the minute parallel tubules of the dentin tissue of a tooth.
  • intrafibrillar collagen mineralization refers to the process in which minerals are incorporated within the gap zone of collagen fibrils, such as mineralization with hydroxyapatite or calcium phosphate, by methods such as those described in the article Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization, Thrivikraman et al., Nature Communications, 10, 3520 (2019).
  • mineralized hydrogels refers to hydrogels treated with crystallizable metals, including alkali metals and/or earth alkali metals, such as ionic calcium and/or ionic phosphorus, to mimic the mineralized microenvironment of native bone.
  • crystallizable metals including alkali metals and/or earth alkali metals, such as ionic calcium and/or ionic phosphorus.
  • nanoscale mineralization refers to preparation of a mineralized matrix, such as a mineralized collagen.
  • collagen membrane refers to a collagenous material that has been compressed to increase its final collagen concentration.
  • collagen membranes can be fabricated from a quantify rat tail type I collagen that was centrifuged at 3500 g for about 20 minutes at 22°C to increase its collagen concentration from about 3.0 mg/mL to about 75 mg/mL.
  • Collagen membranes can be made using a variety of conditions, for example, a temperature range from 4 to 37°C, and a time range from 5 minutes to 24 hours. The centripetal force can range from 3, 000-4, 000g.
  • primary dentin refers the dentin layer that generally forms closest to the enamel of a tooth, and new dentin which has not mineralized.
  • second dentin refers to the dentin layer that forms after the formation of roots in the tooth.
  • tertiary dentin refers to dentin generally formed as a defense mechanism to stimuli, such as caries.
  • Tertiary dentin also referred to in the art as “reparative dentin” or “reactionary dentin” is known by skilled persons to form as a result of trauma to odontoblasts.
  • tertiary dentin-like tissue constructs refers to an engineered tissue product that mimics the characteristics and qualities of native tertiary dentin. Skilled persons will understand that such tissue constructs are useful for restoring, maintaining, or improving native tissue function.
  • tubular recess density refers to the number or count of a tubular recesses per unit of area, such as in an attempt to mimic the tubular structures found in native dentin.
  • a porous compressed collagenous membrane may have a tubular density of about 1 .0 X 10 4 tubular recesses/mm 2 .
  • the tubular density is of from about 1 ,000 tubular recesses /mm 2 to about 10,000 tubular recesses/mm 2 .
  • porous compressed collagenous membranes wherein the tubular density is, respectively, a) from about 1 ,000 tubular recesses/mm 2 to about 50,000 tubular recesses/mm 2 ; b) from about 50,000 tubular recesses/mm 2 to about 100,000 tubular recesses/mm 2 ; c) from about about 25,000 tubular recesses/mm 2 to about 50,000 tubular recesses/mm 2 ; d) from about 50,000 tubular recesses/mm 2 to about 75,000 tubular recesses/mm 2 ; e) from about 25,000 tubular recesses/mm 2 to about 75,000 tubular recesses/mm 2 ; f)) from about 1 ,000 tubular recesses/mm 2 to about 10,000 tubular recesses/mm 2 ; g) from about 10,000 tubular recesses/mm 2 to about 20,000 tubular recesses/mm 2 ; h) from about 20,000 tubular recesses/mm 2 to about 30,000 tubular recesses/mm
  • a compressed collagenous matrix may be produced by applying sufficient centripetal force to a collagenous matrix to express water from the collagenous matrix by compression and thereby increase the volume fraction of the collagenous material relative to that of water.
  • the verb “to mineralize” and all its verb conjugates refer collectively and interchangeably to intra- and extra-fibrillar collagen mineralization.
  • the verb “to compress” and all its verb conjugates refers to the process known in the art of applying an unconfined compressive force to a reconstituted collagen gel (such as a hydrogel) to expel aqueous component from the reconstituted collagen gel, which does not return upon removal of the compressive force (Brown R.A. et al., Ultrarapid Engineering of Biomimetic Materials and Tissues. Adv. Fund. Mater. 15, 1762-1770 (2005)).
  • Plastic compression is used by skilled persons to reduce and thereby tune the aqueous component of a collagenous matrix to mimic that of native tissues, typically from about 99.8 percent by volume of aqueous component to about 85% to 90% v/v (Cheema U. and Brown R.A., Rapid Fabrication of Living Tissue Models by Collagen Plastic Compression: Understanding Three-Dimensional Cell Matrix Repair In Vitro. Advances in Wound Care, Vol. 2; Num. 4 (2013)). Skilled persons will understand that such removal of excess fluid may be achieved by a range of methods, including blotting/capillary action, mechanical compression, centrifugation, and osmotic pressure.
  • plastic compression allows skilled persons to pre-determine the final collagen density of a collagenous matrix through controlling the amount of fluid removed, thereby facilitating the fabrication of a biomimetic collagenous matrix that comprises a concentration of collagen that mimics that of a specific native tissue, such as tertiary dentin.
  • Most hydrogel concentrations reported in the art teach collagen starting concentration fabrication parameters that are generally between .1 % and 10% v/v, which significantly limits their value for mimicking 3D tissues due to their nonphysiological strength and microstructure and results in an inability to support microfabrication as bulk gels (Antoine E.E. et al., Review of Collagen I Hydrogels for Bioengineered Tissue Microenvironments: Characterization of Mechanics, Structure, and Transport. Tissue Eng Part B Rev. Dec 1 ; 20(6): 683-696 (2014).
  • a biomimetic, bioartificial dentin or dentin-like material for promoting regeneration of dentin tissue comprises a mineralized compressed collagenous matrix and a plurality of isolated dentin matrix molecules (DMM).
  • the biomimetic, bioartificial dentin or dentin-like material is referred to herein as “eDentin.”
  • eDentin is also referred to herein as dentin-like material, biomimetic reparative dentin or dentin-like material, and bioartificial dentin or dentin-like material.
  • a biomaterial able to mimic the nanostructure and composition of dentin can represent a “ready-to-use” biocompatible reparative dentin layer, which will work faster and more efficiently than current pulp capping cements including silicate cements. While limited efforts focused on engineering models of dentin in vitro, fabrication of off-the-shelf ready to use dentin-like biomaterials have not been reported.
  • a dentin-like material eDentin
  • eDentin a dentin-like material that may be engineered and characterized with properties matching that of the native tissue and which can be readily applied into the pulp.
  • a bioartificial dentin-like material is described herein, which is useful as a tissue-engineered reparative dentin.
  • This biomaterial can consistently generate thick and scalable tissues that have the potential for rapid repair of damaged tissue and an immediate protective function for the pulp.
  • the nano/microstructural and mineral mimicry of cellularized reparative dentin the biomaterial facilitates microengineering this system with the presence of mineralized dentinal tubules allows control of the process of encapsulating cells in a mineralizing matrix.
  • the encapsulation of cells and biomolecules in high-density mineralized collagen (HDMC) allows for embedding both cells and dentin matrix molecules in high-density mineralized collagen that mimic the materials characteristics of reparative dentinal tissue.
  • HDMC high-density mineralized collagen
  • the collagen in the provided materials and methods provided herein can be a type 1 collagen matrix.
  • the type 1 collagen matrix may be prepared by reconstituting acid solubilized type 1 collagen.
  • the collagen may be at a concentration of about 0.5 mg/mL to about 50.0 mg/mL.
  • collagen examples include, but are not limited to, collagen type II, collagen type III, collagen type IV, collagen type V, collagen type VI, collagen type VI, collagen type VII, collagen type VIII, collagen type IX, collagen type X, collagen type XI, collagen type XII, collagen type XIII, collagen type XIV, collagen type XV, collagen type XVI, collagen type XVII, collagen type XVIII, collagen type XIX, and collagen type XX, or a combination thereof.
  • “Type I collagen” or “Type 1 collagen” refers to the fibrillar-type collagen that is the most abundant form of human collagen and the key structural composition of several tissues.
  • the collagen is a rat tail type I collagen or a human type I collagen.
  • the bioartificial dentin-like material further comprises dispersing the mineralized compressed collagenous matrix and the plurality of isolated dentin matrix molecules (DMM).
  • the DMMs can be present in the reparative dentin material at a concentration of: a) of from about 0.1 pg/ml to about 250 pg/ml; b) of from about 250 pg/ml to about 500 pg/ml; c) of from about 500 pg/ml to about 1000 pg/ml; and d) of from about 1000 pg/ml to about 5000 pg/ml, or e) greater than 5000 pg/ml.
  • the DMMs can comprise dentin matrix proteins having an origin selected from human, ovine, porcine, and bovine.
  • the DMMs can, optionally, comprise human dentin matrix proteins, bovine dentin matrix proteins, or porcine dentin matrix proteins.
  • the bioartificial dentin-like material is crosslinkable.
  • the bioartificial dentin-like material can further comprise a crosslinking agent.
  • the bioartificial dentin-like material is lyophilized.
  • a bulk material comprising a plurality of the reparative dentin material is provided.
  • the provided methods can include the step of adding DMMs to the collagen first followed by applying force to the collagen/DMM mixture to create the high-density collagen/DMM material.
  • the collagen/DMM material can then be contacted with a cross-linking agent.
  • the methods include the steps of applying force to collagen to created a high-density collagen material followed by adding DMM to the collagen material and then contacting the DMM/collagen material with a crosslinking agent.
  • the methods include the steps of applying force to collagen to created a high-density collagen material followed by adding a crosslinking agent to the collagen material and then adding DMM to the collagen material.
  • the reparative dentin material can further comprise cells.
  • the cells can be stem cells selected from any one of: a) dental pulp stem cells (DPSC), b) dental follical progenitor cells (DFPCs), c) stem cells from Apical Papilla (SOAP), d) periodontal ligament stem cells (PDLSCs), e) stem cells from human exfoliated deciduous teeth (SHED), and f) mesenchymal-stem-cell-like (MSC) cells.
  • DPSC dental pulp stem cells
  • DFPCs dental follical progenitor cells
  • SOAP Apical Papilla
  • PDLSCs periodontal ligament stem cells
  • SHED human exfoliated deciduous teeth
  • MSC mesenchymal-stem-cell-like
  • the cells can be present in the material at a concentration selected from any of: of from about 1 .0 x 10 2 cells/mL to about 1.0 x 10 3 ; of from about 1.0 x 10 3 cells/mL to about 1 .0 x 10 4 ; of from about 1 .0 x 10 5 cells/mL to about 1 .0 x 10 6 ; or of from about 1 .0 x 10 6 cells/mL to about 1 .0 x 10 7
  • the bioartificial dentin-like material can be formed into a tertiary dentin-like tissue construct.
  • the dentin-like tissue construct is formed by applying centripetal force to a quantity of the bioartificial dentin-like material.
  • centripetal force is applied to the quantity of the material for about 20 minutes at about 22°C to thereby fabricate a mineralized compressed collagenous matrix.
  • the bioartificial dentin-like material can comprise a set of tubular recesses, each tubular recess in the set extending from the surface of, and into, the dentin-like tissue construct of from about 0.5 pm to about 10 pm of depth to form a porous compressed collagenous membrane having a tubular recess density.
  • the tubular recess density of the porous compressed collagenous membrane can be: a) of from about 1 .0 x 10 3 tubular recesses/mm 2 to about 1 .0 x 10 5 tubular recesses/mm 2 , b) of from about 1 ,000 tubular recesses /mm 2 to about 10,000 tubular recesses/mm 2 ; c) of from about 1 ,000 tubular recesses/mm 2 to about 50,000 tubular recesses/mm 2 ; d) of from about 50,000 tubular recesses/mm 2 to about 100,000 tubular recesses/mm 2 ; e) of from about 25,000 tubular recesses/mm 2 to about 50,000 tubular recesses/mm 2 ; f) of from about 50,000 tubular recesses/mm 2 to about 75,000 tubular recesses/mm 2 ; g) of from about 25,000 tubular recesses/mm 2 to about 75,000 tubular recesses/mm 2 ; h) of from about 1 ,000 tub
  • a silicon wafer can be used as a positive mold with 2 um-pillar micropatterns via photolithography at a density of between 10 4 to 10 5 pilars/mm 2 .
  • the wafer has micropatterns at a density of 10 ⁇ pilars/mm ⁇ .
  • High-density collagen membranes can be prepared, dispensed on top of the silicon wafer as a positive mold with 2 um- pillar micropatterns via photolithography at a density of 10 ⁇ pilars/mm ⁇ , allowed to assemble at 37°C and made into a thickness 0.1-0.6 mm.
  • the bioartificial dentin-like material can be configured as a therapeutic compound for administering to a subject in need.
  • a method of making compressed collagenous matrix comprises providing a quantity of collagen matrix allowed to self-assemble.
  • the method includes allowing the collagen matrix to self-assemble at about 37°C for about 15 minutes and applying about 3500 g of centripetal force to the quantity of collagen matrix for about 20 minutes at about 22°C to thereby fabricate a compressed collagenous matrix.
  • the collagen matric may be allowed to self-assemble of from about 35°C to about 39°C.
  • the about 3500 g of centripetal force may be applied to the quantity of collagen matrix of from about 20°C to about 24°C.
  • Collagen matrix can also be made using a variety of conditions, for example, a temperature range from 4 to 37°C, and a time range from 5 minutes to 24 hours. The centripetal force can range from 3,000- 4,000g.
  • the cell media can contain, by way of example, alpha-MEM with 100 pg/mL of milk-derived osteopontin (mOPN), about 4.5 mM CaCI2, and about 2.1 mM K2HPO4, buffered in HEPES, for about 72 hours in a shaker at about 37°C in about 100% humidity.
  • mOPN milk-derived osteopontin
  • the cell media contains a nucleation inhibitor.
  • Suitable nucleation inhibitors include, but are not limited to, Osteopontin, Osteocalcin, Osteonectin, bone sialoprotein, dentine phosphoryn, dentin matrix protein 1 , dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein, chondrocalcin, proline-rich proteins such as Proline-rich protein 1 , Proline-rich protein 2, and Proline-rich proteins, PRP1-T1 , PRP3-T1 , Histatin 5, MG1 , MG2, Asialo_MG2, Amylase, statherin, cystatin S, cystatin SN, Cystatin S1, fetuin, HSA, poly-L-aspartic acid, or combinations thereof.
  • the osteopontin concentration may be from about 50 pg/mL to about 150 pg/mL.
  • One embodiment provides a method for mineralizing a biomimetic, bioartificial dentin-like material comprising incubating the plastically compressed collagenous matrix into a cell media comprising from about 90-110 pg/ml poly-L-aspartic acid (27 kDa).
  • Another embodiment provides a method using a cell media comprising from about 90-110 pg/ml poly-L-aspartic acid (27 kDa) and from about 4.0 to about 5.0 mM calcium ions (from the list of calcium salts described herein).
  • a further embodiment provides a method using a cell media comprising from about 90-110 pg/ml poly-L-aspartic acid (27 kDa), from about 4.0 to about 5.0 mM calcium ions, and from about 1 .8-2.5 mM phosphate ion.
  • the cell media further comprises 80-120 pg/mL osteopontin.
  • glutaraldehyde 200 pL of 5% Glutaraldehyde can be added to collagen and allowed to incubate for 1 hour at room temperature while shaken. Samples can be washed as needed, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10 or more times. For example, samples can be washed with PBS.
  • a method of making a porous compressed collagenous membrane comprises providing a compressed collagenous matrix; combining a set of DMMs and the compressed collagenous matrix within a polymer material to make a hydrogel composition; mineralizing the hydrogel composition; dispersing the hydrogel composition onto a shaping device, the shaping device being configured as a positive mold comprising a set of pillars having a pillar density; and removing the hydrogel composition from the shaping device.
  • the shaping device can be a silicon wafer or a microfluidic channel.
  • the set of pillars is formed via photolithography.
  • Each pillar in the set of pillars can have a height selected from: a) of from about 1 pm to about 2 pm; b) of from about 2 pm to about 3 pm; c) of from about 3 pm to about 4 pm; d) of from about 4 pm to about 5 pm; or, e) of from about 5 pm to about 10 pm.
  • the pillar density is selected from any of: a) of from about 1 .0 x 10 3 pillars/mm 2 to about 1 .0 x 10 5 pillars/mm 2 , b) of from about 1 ,000 pillars/mm 2 to about 10,000 pillars/mm 2 ; c) of from about 1 ,000 pillars/mm 2 to about 50,000 pillars/mm 2 ; d) of from about 50,000 pillars/mm 2 to about 100,000 pillars/mm 2 ; e) of from about 25,000 tubular pillars/mm 2 to about 50,000 pillars/mm 2 ; f) of from about 50,000 pillars/mm 2 to about 75,000 pillars/mm 2 ; g) of from about 25,000 pillars/mm 2 to about 75,000 pillars/mm 2 ; h) of from about 1 ,000 pillars/mm 2 to about 10,000 pillars/mm 2 ; i) of from about 10,000 pillars/mm 2 to about
  • the porous compressed collagenous membrane can be chopped and frozen until further use.
  • the porous compressed collagenous membrane is lyophilized.
  • a method of promoting regeneration of dental tissue comprising delivering the reparative dentin material to a surface of a dental tissue.
  • a method of promoting regeneration of dental tissue comprises delivering the reparative dentin material to a surface of a tooth.
  • composition for promoting regeneration of dental tissue comprises a dentin matrix molecules (DMM) dispersed within a collagen.
  • DDM dentin matrix molecules
  • the DMMs can be obtained from harvested dentin and may include one or more compounds selected to stimulate cellular invasion promoting tissue regeneration in the pulp of a tooth.
  • the DMM comprises one or more dentin matrix proteins (DMPs).
  • DMPs dentin matrix proteins
  • the particular compound or compounds provided by the DMM may depend upon the nature of the dentin from which the material is sourced.
  • the DMM can comprise one or more human DMPs.
  • the DMM comprises one or more bovine DMPs.
  • the DMMs can be present in the reparative dentin material in a concentration of from about 100 pg/ml to about 1 ,200 pg/ml.
  • the concentration is from about 250 pg/ml to about 600 pg/ml.
  • the polymer material may be self-curing and/or may be curable by other means, such as by light, temperature, chemical agents, or mechanical means.
  • the composition further comprises a crosslinking agent selected to facilitate crosslinking of the polymer material.
  • the crosslinking agent is glutaraldehyde, genipin, or ruthenium.
  • Other suitable crosslinking agents include, but are not limited to, Ethyl-3-[3-dimethylaminopropyl] Carbodiimide Hydrochloride (EDO), Tannic acid, Hesperidin, Riboflavin and the Proanthocyanidin-rich cross linkers, Cathechin, Epicathechin, Epillocathechin, and Epillocathechin gallate.
  • the composition can comprise a polymer material that is at least partially crosslinked to provide a hydrogel material containing DMMs.
  • the hydrogel material may be disaggregated into discrete particles or pieces.
  • the hydrogel material may be lyophilized or freeze-dried to provide a particulate material that can be rehydrated to enable release of DMMs from the material.
  • a method for making a composition as described herein can comprise providing DMMs and combining the DMMs with a polymer material such as methacylated gelatin, methacylated hyaluronic acid, alginate, agarose, chitosan, PEGDA, or PEGDMA, that is crosslinkable, biocompatible and biodegradable. These can be further combined with a crosslinking agent such as the cross-linking agents, glutaldehyde, genipin, or ruthenium.
  • a polymer material such as methacylated gelatin, methacylated hyaluronic acid, alginate, agarose, chitosan, PEGDA, or PEGDMA, that is crosslinkable, biocompatible and biodegradable.
  • a crosslinking agent such as the cross-linking agents, glutaldehyde, genipin, or ruthenium.
  • crosslinking agents include, but are not limited to, Ethyl-3-[3-dimethylaminopropyl] Carbodiimide Hydrochloride (EDC), Tannic acid, Hesperidin, Riboflavin and the Proanthocyanidin-rich cross linkers, Cathechin, Epicathechin, Epillocathechin, and Epillocathechin gallate.
  • EDC Ethyl-3-[3-dimethylaminopropyl] Carbodiimide Hydrochloride
  • Tannic acid Tannic acid
  • Hesperidin Riboflavin
  • Proanthocyanidin-rich cross linkers Cathechin, Epicathechin, Epillocathechin, and Epillocathechin gallate.
  • the method can further comprise at least partially crosslinking the polymer material to produce a hydrogel.
  • Providing DMMs can comprise obtaining dentin, for example from harvested teeth.
  • the dentin may be obtained from teeth of one or more origins including but not limited to, human, bovine, porcine and ovine.
  • the dentin may then be processed to extract and isolate DMMs including DMPs from the dentin.
  • the DMMs may be stored for a time before use. This can comprise lyophilizing the DMMs to produce a powder that may be stably stored at low temperature.
  • the DMMs may be combined with a crosslinkable polymer material so that the DMMs are dispersed in the polymer material.
  • the amount of DMMs are selected to provide a concentration of DMMs in the composition from about 100 pg/ml to about 1 ,200 pg/ml. In a more specific embodiment, the concentration is from about 250 pg/ml to about 600 pg/ml.
  • the DMMs and the polymer material may additionally be combined with a crosslinking agent.
  • the polymer material can comprise the crosslinking agent.
  • the crosslinking agent is glutaraldehyde, genipin, or ruthenium.
  • Other suitable crosslinking agents include, but are not limited to, Ethyl-3-[3-dimethylaminopropyl] Carbodiimide Hydrochloride (EDC), Tannic acid, Hesperidin, Riboflavin and the Proanthocyanidin-rich cross linkers, Cathechin, Epicathechin, Epillocathechin, and Epillocathechin gallate.
  • the method can further comprise curing the composition to produce a hydrogel material by inducing crosslinking of the polymer material.
  • the resulting material may be frozen until use without lyophilization or the resulting material may be lyophilized to enhance its stability during storage. Lyophilization can also provide greater ease of handling the bulk material. In some embodiments, lyophilization is carried out by subjecting the material to a gradual freezing rate between 0.5° C./min to 5° C./min. The resulting lyophilized material can also be subsequently subjected to a sterilization process. Such a process may include, for example, the application of gamma-radiation to the lyophilized product, or other approaches suitable for use on such material without affecting its stability.
  • the lyophilized material may be stored at low temperature, such as at or below about -10°C, until use.
  • the method can comprise delivering a composition to a cavity in a tooth, where said composition comprises DMMs.
  • the composition can further comprise a polymer material, and can further include a crosslinking agent.
  • the polymer material can comprise a collagen.
  • the crosslinking agent is a chemical crosslinking agent.
  • the method may comprise delivering the composition as a membrane to the surface of the tooth or directly in contact with the dental pulp.
  • the membrane material is lyophilized and is rehydrated after delivery, which may occur passively by the moisture present in the tooth cavity and/or be accomplished by addition of liquid.
  • the applied composition is applied directly to the surface of the tooth or the dental pulp.
  • the composition is applied with an adhesive to the surface of the tooth or the dental pulp.
  • Also provided is a method for preparing a biomimetic, bioartificial dentin material by treating a compressed collagenous matrix with an effective media containing a) one or more dentin matrix proteins; b) one or more calcium salts; c) one or more phosphate salts or any combination thereof for a time and at a temperature effective to produce the biomimetic, bioartificial dentin material.
  • the one or more dentin matrix proteins in the effective media in the method above can be dentin matrix acidic phosphoprotein 1 (DMP1 ), dentin phosphophoryn (DMP2), dentin matrix protein 4 (DMP4 or FAM20C), dentin sialophosphoprotein (DSP), bone sialoprotein, osteopontin, osteonectin, osteocalcin, decorin, and matrix extracellular phosphoglycoprotein (MEPE).
  • the effective media comprises a non-collagenous protein selected from the group of Osteopontin, dentin matrix protein 1 , bone sialoprotein, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein.
  • the effective media comprises Osteopontin.
  • the effective media can comprise a calcium salt selected from the group of calcium chloride (CaCI2), calcium chloride dihydrate, calcium fluoride (CaF2), calcium bromide (CaBr2), calcium carbonate, magnesium-enriched calcium carbonate, and calcium nitrate (Ca(NO3)2).
  • the effective media comprises calcium chloride.
  • the calcium salt and/or the phosphate salt may comprise one or more of the compounds or materials selected from the group of calcium phosphate, calcium- deficient apatite, hydroxyapatite, amorphous calcium phosphate, dicalcium phosphate, octocalcium phosphate, tricalcium phosphate, tetracalcium phosphate, and calcium phosphate hydroxide.
  • Phosphate salts useful in the effective media include those selected from the group of calcium phosphate, potassium phosphate (K2PO4), sodium dihydrogen phosphate (Na2HPO4), (NH4)2HP04, and dicalcium phosphate dihydrate.
  • the phosphate salt can be calcium phosphate or potassium phosphate.
  • the phosphate salt comprises potassium phosphate.
  • the effective media in the method for preparing a biomimetic, bioartificial dentin-like material can further comprise a buffer, such as, for example, HEPES, Tris buffer, phosphate buffer, goods buffers, simulated body fluid (SBF), and the like.
  • a buffer such as, for example, HEPES, Tris buffer, phosphate buffer, goods buffers, simulated body fluid (SBF), and the like.
  • a Also provided is a method of making a compressed collagenous matrix comprising the steps of: a) treating a quantity of collagen matrix at a temperature and for a time that allow it to self-assemble, and b) providing a selfassembled collagen matrix; and applying to the self-assembled collagen matrix a centripetal force to thereby fabricate the compressed collagenous matrix.
  • the collagen matrix in step a), above is treated with a temperature of from about 4°C to about 37°C, e.g., 15°C to about 30°C, for a time that allow it to self-assemble, providing a self-assembled collagen matrix.
  • the collagen matrix in step a), above is treated at a temperature of from about 18°C to about 28°C for a time that allow it to self-assemble, providing a self-assembled collagen matrix.
  • the collagen matrix in step a), above is treated at a temperature of from about 20°C to about 24°C for a time that allow it to self-assemble, providing a self-assembled collagen matrix.
  • the collagen matrix is treated at a temperature of about 22°C for a time that allow it to self-assemble.
  • the method further includes treating the collagen matrix to a centripetal force of from about 3,000 g to about 4,000 g to provide the self-assembled collagen matrix.
  • the collagen matrix in step a) is treated with the temperature indicated herein and further treated to a centripetal force of from about 3,200 g to about 3,800 g.
  • the collagen matrix in step a) is treated with the temperature indicated herein and further treated to a centripetal force of from about 3,400 g to about 3,600 g.
  • the collagen matrix in step a) is treated with the temperature indicated herein and further treated to a centripetal force of from about 3,450 g to about 3,550 g.
  • the collagen matrix in step a) can be treated with the temperature indicated herein and further treated to a centripetal force of about 3,500 g to provide the self-assembled collagen matrix.
  • the time for self-assemble can be from 5 minutes to 24 hours.
  • the collagen matrix in step a) can be treated with the temperature and/or centripetal force indicated herein for a time of from about 5 minutes to about 30 minutes to provide the self-assembled collagen matrix of interest.
  • the collagen matrix in step a) can be treated with the temperature and/or centripetal force indicated herein for a time of from about 10 minutes to about 20 minutes to provide the self-assembled collagen matrix of interest.
  • the collagen matrix in step a) can be treated with the temperature and/or centripetal force indicated herein for a time of from about 12 minutes to about 18 minutes to provide the self-assembled collagen matrix of interest.
  • the collagen matrix in step a) is treated with the temperature and/or centripetal force indicated herein for a time of: i) from about 10 minutes to about 15 minutes; ii) from about 15 minutes to about 20 minutes; iii) from about 12 minutes to about 18 minutes; and for about 15 minutes to provide the self-assembled collagen matrix of interest.
  • eDentin can be used in the clinic or a clinical setting.
  • the lyophilized eDentin material can be rehydrated when you put it in contact with the pulp. Alternatively, you can use a fluid to rehydrate it. In the clinic, you would either place the lyophilized eDentin material on the surface of the tooth or placed on exposed pulp at the site of repair.
  • the eDentin material can be applied with or without an adhesive.
  • Suitable adhesives include, but are not limited to 10-MDP (10-Methacryloyloxydecyl dihydrogen phosphate) and a hydrophilic photoinitiator, which includes 2-hydroxy-1- [4- (2-hydroxyethoxy) phenyl]-2-methyl-1 -propanone (Irgacure 2595) and the water- soluble derivatives of acylphosphine oxides, e.g., monoacylphosphine oxide (MAPO) and bisacylphosphine oxide (BAPO) and Lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP).
  • 10-MDP 10-Methacryloyloxydecyl dihydrogen phosphate
  • a hydrophilic photoinitiator which includes 2-hydroxy-1- [4- (2-hydroxyethoxy) phenyl]-2-methyl-1 -propanone (Irgacure 2595) and the water- soluble derivatives of acylphos
  • lyophilized eDentin material When used with an adhesive, lyophilized eDentin material can first be placed at the site of repair (e.g. exposed pulp) then an adhesive containing 10-MDP and/or hydrophilic photoinitiator can be applied over the eDentin surface. Alternatively. eDentin is placed at the sight of the repair (e.g. exposed pulp) then a glass ionomer cement is used.
  • Figure 12 detailed in the example below provides an exemplary way of using eDentin in the clinic.
  • reactionary dentinogenesis represents an upregulation of existing primary odontoblasts from their quiescent state
  • reparative dentinogenesis is a far more complex process, requiring the recruitment and differentiation of dental pulp stem cells (DPSC) into a secretory odontoblast-like phenotype.
  • DPSC dental pulp stem cells
  • these cells can be entrapped within the mineralized structure of the tertiary dentin, giving rise to a morphology typically referred to as ‘osteo-dentin’.
  • DPSC migration and differentiation require the presence of active molecules to direct this process.
  • the biomolecules that are fossilized within the dentin matrix are solubilized upon demineralization and/or matrix degradation of dentin, allowing them to diffuse down the dentinal tubules to the pulp tissue and recruiting the stem cells.
  • the presence of bioactive DMMs embedded in a collagenous matrix, and the existence of cells embedded in the osteo-dentin are two hallmarks of the native reparative dentin that can be controllably replicated in our biomaterial.
  • Many dental materials for vital pulp therapy such as silicate cements (i.e. MTA) have sought to leverage the ability of the dental pulp to 'protect itself' by inducing the solubilization of dentin matrix molecules.
  • DMMs non-collagenous dentin matrix molecules
  • DM Ms non-collagenous dentin matrix molecules
  • Isolated DMMs can be stored (-80°C) for more than 5 years without losing potency, which makes them ideal candidates for use in regenerative dental materials.
  • mineral trioxide aggregate causes severe inflammation, while eDentin is a 100% biocompatible and non-inflammatory, ready-to-use membrane that is placed onto the dental pulp (like a “band-aid”) releasing dentin matrix molecules (DMMs) to attract pulp cells that will secrete fresh calcified matrix, thus integrating eDentin into the pulp — dentin complex.
  • DMMs dentin matrix molecules
  • Ultramicrotome-sectioned TEM images may be analyzed with the respective selective area electron diffraction (SAED) spectra, and quantitative backscattered SEM reconstructions to further evaluate intrafibrillar collagen mineralization which allows for quantification of the percentage of nanoscale mineralization in 3D for nanoscale mineralized hydrogels.
  • Figures 2A to 2G are images and bar graphs showing SEM and TEM of eDentin’s nanostructure vs. native calcified collagen fibril (FIGS. 2A-2D); mineralization, composition, and elastic modulus of eDentin vs. pure collagen and native mineralization tissue controls (FIGS. 2E-2G).
  • FIG. 5B histological sections of MTA samples showing tissue disorganization, and severe inflammation (arrows), while eDentin (FIG. 5C, 5D - asterisk) is not inflammatory, can be immediately juxtaposed with the pulp tissue (FIG. 5D - dotted line) as a ready-to-use mineralized engineered dentin (i); H&E and von Kossa.
  • Figures 6A to 6B shows evaluation regarding the order of use of the crosslinker relative to mineralization.
  • Figures 6A to 6B show that mineralization can be performed either prior to or following crosslinking of the collagen.
  • FIG. 6A and FIG. 6B show better mineralization is achieved prior to crosslinking as evidenced by higher mineral to matrix ratio and crystallinity index from FTIR data as well as the more opaque appearance in photographs.
  • FIG. 7A shows scanning Electron Microscope (SEM) images demonstrate the surface morphology with different crosslinkers appear comparable.
  • FIG. 7B shows cell viability appear comparable using different crosslinking agents.
  • SEM scanning Electron Microscope
  • FIG. 8A to 8G shows the characterization of mineralized collagen.
  • FIG. 8E EDX spectra of high-density mineralized samples confirmed the presence of Ca and P.
  • FIG. 8F minerakmatrix ratio and
  • FIG. 8G crystallinity index of high-density mineralized and non-mineralized samples and
  • DMMs dentin matrix molecules
  • FIG. 4B tertiary dentin formation using engineered hydrogels supplemented with DMMs
  • FIG. 4C MTA induced inflammation prior to tertiary dentin formation in vivo.
  • DMMs were loaded into the constructs after eDentin mineralization to prevent unforeseen effects on the calcification process.
  • mineralized cell-loaded membranes were mineralized then immersed for 24 hours in cell medium containing DMMs to allow the DMMs to adsorb onto the porosity of material overnight at 4°C.
  • DMM passive release was monitored daily from eDentin and dentin disks into 0.1 % (w/v) BSA in PBS for seven days, by collecting the supernatant daily and measuring the protein content with a BCA kit.
  • Figure 9 shows DMM release kinetics confirms that DMMs are incorporated and released from eDentin.
  • the DMM release protein assay was done in different timepoints (1 h, 12h, 1 day, 2 days and 7 days) using BCA Protein Assay Kit. Further aliquots of the supernatant may be inspected via ELISA for growth factors that we have characterized to be abundant in the dentin matrix (TGFp, VEGF, IGF).
  • FIG. 10A shows eDentin (compressed, mineralized high-density fibrillar collagen with DMM, abbreviated cm-HDFC + DMM) supports better mineral deposition by odontoblast like cells (OD-21 cells) compared to high-density fibrillar collagen (HDFC) alone using a Alizarin red S stain.
  • FIG. 10A imagens
  • FIG. 10B Quantification of Alizarin red S stain.
  • FIG. 11A Scanning electron microscope image showing that eDentin is able to bond to formulated adhesive containing Methacryloyloxydecyl dihydrogen phosphate (MDP) and hydrophilic photoinitiator.
  • MDP Methacryloyloxydecyl dihydrogen phosphate
  • FIG. 11 B 10-MDP and hydrophilic photoinitiator after shear stress. The collagen is present on the surface suggesting a bond between the adhesive and the membrane.
  • FIG. 11 C Adhesive single bond plus after sheer stress. There is less collagen present on the surface suggesting the adhesion is not good.
  • Figure 12 is a schematic showing an exemplary application of eDentin in pulp capping procedures.
  • An adhesive containing 10-MDP and hydrophilic photoinitiator can be applied over the eDentin surface as shown in D.
  • the 10-MDP monomer is capable of chemically bonding to calcium present in the membrane as shown in D. and the resin matrix in the restorative material as shown in E.
  • Hydrophilic photoinitiator is important to promove the monomer conversion in a wet surface (look the last figure).
  • the application mode of eDentin for glass ionomer is after cavity removal the eDentin must be inserted over the exposed pulp. After eDentin application, the glass ionomer can be applied according to the manufacture instructions.

Abstract

La présente divulgation concerne des compositions, des kits et des méthodes destinés à favoriser la régénération de tissus dentaires, en particulier de tissu de dentine et de pulpe dentaire.
PCT/US2023/031056 2022-08-24 2023-08-24 Matériau de dentine biomimétique et réparateur pour favoriser la régénération de tissu de dentine et procédés associés WO2024044319A2 (fr)

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